Internal Tandem Duplication Mutation Of FLT3 (FLT3/ITD) Induces Increased Homologous Recombination DNA Repair Activity, Drug Resistance and Sister Chromatid Exchanges In Acute Myeloid Leukaemia (AML)

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It is has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.

To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through production of excessive DNA double strand breaks (DSB) and up-regulation of HR DNA repair. Primary NK-AML (n=17) were initially characterised by SNP-A and DNA sequencing. 12/17 primary NK-AML had FLT3/ITD mutations with a clone size ≥ 25%, but were wild type (WT) for RAS. From this cohort 6/12 possessed CN-LOH with 3/12 having CN-LOH at 13q. 5/17 primary NK-AML were WT for FLT3 and RAS, but had nucleophosmin (NPM) and DNA methyltransferase 1 (DNMT1) mutations. From this cohort, 1/5 primary NK-AML + WT FLT3 had CN-LOH.

We showed that primary AML + FLT3/ITD cohort, the MOLM 13 cell line that possesses a FLT3/ITD mutation and WT FLT3 KG-1 cells transfected with a FLT3-ITD expression vector demonstrate increased ROS compared to the WT FLT3 primary NK-AML cohort, the WT FLT3 cell line, U937 and KG-1 transfected with empty vector. Immunofluorescence studies showed that primary AML with FLT3/ITD, MOLM 13 and FLT3-ITD transfected KG-1 cells have increased mobilization of phospho-γH2AX (a DNA DSB intermediate) and RAD51 (a HR factor) to sites of DNA DSB in response to mitomycin C (MMC) induced DNA damage compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (phospho-γH2AX, 29% vs. 21%, p<0.005 and RAD51, 25% VS 19%, p<0.01). Moreover, we show that HR is augmented in FLT3-ITD transfected cells and MOLM-13 with and without exogenous DNA damage using DR-GFP gene reporter assays. Increased HR activity has been associated with increased resistance to genotoxic agents. Using soft agar clonogenic assays, MOLM 13 and FLT3-ITD transfected KG-1 showed considerably increased resistance to MMC and the radiomimetic agent, bleomycin compared to U937 cells and KG-1 + empty vector. Finally, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome crossing over. FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells and KG-1 transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than non-13q CN-LOH primary AML + FLT3-ITD mutation (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored ROS levels, HR activity, sensitivity to MMC and SCE to levels observed for WT FLT3. These findings not only provide a strong proof of principle that FLT3-ITD derived DNA damage is the trigger for CN-LOH in NK-AML, but further our understanding of leukaemic progression and relapse.